Lactobacillus formulations with improved stability and efficacy

ABSTRACT

A composition is provided herein directed to a mixture of one or more  Lactobacillus  sp. and a phenolic compound. In a specific embodiment, disclosed is a  Lactobacillus /phenol mixture that has been lyophilized to form a  Lactobacillus /phenol lyophilized mixture. The  Lactobacillus /phenol mixture of  Lactobacillus /phenol lyophilized mixture may be packaged in a capsule, emulsion, or tablet such that the  Lactobacillus  are protected in a gastrointestinal tract for transport to an intestine of a subject. In another embodiment, a method for treating indigestion, abdominal pain or cephalic syndrome in a subject including orally administering to the subject a composition comprising an effective amount of  Lactobacillus /phenol mixture or lyophilized mixture. In still a further embodiment, of improving immune function in a subject, comprising administering to the subject, the composition comprising an effective amount of  Lactobacillus /phenol mixture or lyophilized mixture.

GOVERNMENT SUPPORT STATEMENT

This invention was made with government support under Grant No.2015-67017-23182 awarded by United States Department of Agriculture. Thegovernment has certain rights in the invention.

BACKGROUND

Type 1 diabetes is an autoimmune disease that affects blood sugarregulation. In type-1 diabetes, a person's immune system makesantibodies that destroy the insulin-producing islet beta cells in thepancreas. As a result, the pancreas fails to make insulin. Withoutinsulin, blood sugar increases and cannot be delivered to the musclesand brain where it is needed. Over time, high blood sugar can lead to anumber of complications including kidney, nerve, and eye damage, andcardiovascular disease. Moreover, cells do not receive the glucosenecessary for energy and normal function. Because people with type 1diabetes can no longer produce their own insulin, they must inject dosesof insulin. They must match the amount of insulin they inject with theirdiet. Keeping blood sugar in a normal, healthy range (what doctors call“good glycemic control”) is the key to preventing long-termdeteriorating effects, including as heart disease, diabetic neuropathy,kidney disease, or poor wound healing resulting from high blood glucoselevels over a prolonged time period.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 a -c. L. johnsonii N6.2 growth and survival during stationaryphase is improved by blueberry aqueous extract under microaerophilic andaerobic conditions. (a) The growth of L. johnsonii N6.2 was assessedunder microaerophilic (filled circles) or aerobic conditions (opencircles). In the samples collected at different time points (0, 6, 12,24 and 36 h), the CFU/ml (solid line) and H₂O₂ production (dashed line)were determined. (b) The effect of increasing concentrations of BAE wastested by growing L. johnsonii N6.2 in MNS-GF (circles), MNS-BB1(diamonds), MNS-BB5 (squares) or MNS-BB10 (triangles) undermicroaerophilic conditions (0 rpm). (c) The effect of the BAE (MNS-BB10)was tested on the growth of L. johnsonii N6.2 under microaerophilic(filled triangles) or aerobic conditions (open triangles). CFU/mL (solidline) and H₂O₂ production (dashed line) were determined under theseconditions. Values shown are the average and SD of experimentaltriplicates.

FIG. 2 a-d . Blueberry aqueous extract increased the resistance of L.johnsonii N6.2 to freeze-drying and improved its survival to storage.(a) Cells were grown for 24 h in MNS-GF or MNS-BB10 under staticconditions, washed twice in PBS buffer, resuspended in 1 ml aliquots inPBS and subjected to freeze-drying and (b) stored at 4° C. for 21 weeks:MNS-GF (circle) and MNS-10BB (triangle). (c-d) In addition, cells grownin MNS-GF for 24 h were resuspended in PBS or increasing concentrationsof BAE: PBS (circle), PBS-BB1 (diamonds), PBS-BB5 (square) and PBS-BB10(triangle). The survival of L. johnsonii N6.2 to freeze-drying (c) andduring storage of the lyophilized powder at 4° C. (d) was determined.CFU/ml were determined before freezing (black bars), after freezing(dark grey bars) and after freeze-drying (light grey bars). Values shownare the average and standard deviations of experimental triplicates.

FIG. 3 a-b . The addition of blueberry aqueous extract to L. johnsoniiN6.2 cells grown under aerobic conditions increase the resistance tofreeze-drying and improved its survival to storage. (a-b) Cells weregrown for 24 h in MNS-GF or MNS-BB10 under aerobic conditions, washedtwice in PBS buffer, resuspended in either 1 ml aliquots in PBS orPBS-BB10, as indicated on the bottom of the figure. (a) Cells weresubjected to freeze-drying and the survival of L. johnsonii N6.2 wasquantified before freezing (black bars), after freezing (dark grey bars)and after freeze-drying (light grey bars). In b, survival was determinedduring storage of the lyophilized powder at 4° C. for 21 weeks:MNS-GF/PBS (circle), MNS-BB10/PBS (square), MNS-GF/PBS-BB10 (triangle)and MNS-BB10/PBS-BB10 (diamond). Values shown are the average andstandard deviations of experimental triplicates.

FIG. 4 a-d . Blueberry aqueous extract maintains cell viability andfunctions as a preservative during lyophilization. Cells grown for 24 hin MNS-GF (a-b) or MNS-BB10 (c-d), were resuspended in PBS, PBS-BB10,PBS-milk and PBS-WPC/SA and subjected to freeze-drying. In a and c,survival of L. johnsonii N6.2 was quantified before freezing (blackbars), after freezing (dark grey bars) and after freeze-drying (lightgrey bars). In b and d, survival was determined during storage of thelyophilized powder at 4° C. for 21 weeks: PBS (circle), PBS-BB10(triangle), PBS-milk (square) and PBS-WPC/SA (diamond). Values shown arethe average and standard deviations of experimental triplicates.

DETAILED DESCRIPTION

The administration of L. johnsonii has been shown to provide remarkablebeneficial effects, particularly in the treatment and prevention of Type1 diabetes and improving immune functions. See U.S. Pat. No. 9,474,773.Provided herein are new methods of formulating L. johnsonii to increaseits bioavailability and effectiveness. It has been discovered thatcombining a phytophenol compound with L. johnsonii acts not only as alyophilizing protectant for L. johnsonii, but also acts to increaseyield and reduce H₂O₂ production in aerobic conditions.

The data provided in the Examples section below relates to testing aphenolic compound containing composition obtained from blueberries. Theresults indicate that the release of free-phenol compounds from theblueberry extract may have a two-fold benefit on L. johnsonii N6.2survival by: (i) increasing the radical scavenging activity to decreasethe overall oxidative stress and (ii) acting as signaling molecules thatmay modulate the expression of genes related to the production of H₂O₂.

According to one embodiment, a composition is provided comprising aLactobacillus sp. combined with a phenolic compound. In a specificexample, the Lactobacillus sp. is L. johnsonii and the phenolic compoundis from an extract of a plant source (i.e. plant extract). Thecomposition may be formulated by methods known to those skilled in theart. In one embodiment, the composition is packaged as a capsule,suppository, tablet, suspension or microemulsion.

Another embodiment pertains to a method of making a compositioncomprising a Lactobacillus sp. The composition comprises admixingLactobacillus sp with a phenolic compound to form a Lactobacillus/phenolmixture. The method may further comprise lyophilizing theLactobacillus/phenol mixture to produce a Lactobacillus/phenollyophilized mixture.

Another embodiment pertains to administering the Lactobacillus/phenollyophilized mixture to a subject in need. A subject in need may be ahuman or non-human mammal in a pre-diabetic or diabetic state or asubject at risk for diabetes (e.g. genetically predisposed to diabetes).

In a further embodiment, composition is administered prior to the onsetof clinical manifestation of type 1 diabetes. The time of administrationis preferably before extensive irreversible beta cell destruction asevidenced by, for example, the clinical onset of type 1 diabetes.Consequently, in at least one embodiment, treatment is administered to asubject in a pre-diabetic state. In at least another embodiment,treatment is administered to a subject at risk for diabetes. In anotherembodiment, treatment is administered to a subject in a diabetic stateto deter or prevent further damage to the HPB cells in the subject.

In addition, a method is provided wherein the subject exhibits acytokine-induced pro-inflammatory response, the composition inhibitsapoptosis of beta cells.

In yet further embodiments, methods for improving immune function in asubject are provided, including administering to the subject, aLactobacillus/phenol mixture. The method may further includeadministering the composition to increase expression of Toll likereceptor 7 (TLR7) and Toll like receptor 9 (TLR9) in the subject.

Overview

Commensal microorganisms such as Lactobacillus, have been used fordecades as food supplements due to its probiotic and/or itsbiotechnological properties (see Kechagia et al. 2013; Klaenhammer etal. 2012; Nya 2015; Shiby and Mishra 2017 for reviews). The Lactobacilligroup is described as fastidious-growing due to the large amount ofnutrients required for growth. The need of expensive components in mediaformulations such as amino acids, vitamins and cofactors withcontrasting poor biomass yields prevent the possibility of using, at anindustrial scale, strains with outstanding probiotic properties. Theseconstraints are furthermore aggravated during the production stage. Thestarter cultures are exposed to conditions such as heat, freezing,freeze-drying and oxidative stress, diminishing overall cell viability.This is a consequence of bacterial metabolites produced during growth(i.e. H₂O₂), mechanical conditions (i.e. shear stress effects) andmethods of preservation and storage (i.e. food stabilizing agents). Themechanisms used by Lactobacillus species to withstand naturalenvironmental stressors (i.e. lactic acid stress, osmotic and oxidativestress) are quite broad (Bravo-ferrada et al. 2015; Lorca and Valdez2001). Several studies have indicated that pre-exposure to one of thesestresses improves survival to different pressures (van de Guchte et al.2002). In L. acidophilus, the mechanisms triggered to resist the lacticacid stress during the stationary phase of growth resulted in crosstolerance to freeze, lyophilization, and oxidative stress (Lorca andValdez 2001).

Certainly, these natural adaptive responses to stress are regularly usedat industrial level, frequently in combination with the addition ofchemical components as preservatives (Endo et al. 2014; Saarela et al.2006). For example, milk and whey are extensively used as carriers forlyophilization and/or spray-drying (Shiby and Mishra 2017; Zheng et al.2015). Alternative matrices are currently being explored in probioticswhich includes the use of fruit or seeds as carriers during theprocessing steps (Bhat et al. 2015; Bustamante et al. 2017; Rajam et al.2015). Many new species/strains of Lactobacillus are currentlyundergoing clinical studies to determine if the probiotic propertiesidentified in vitro/in vivo (cell lines cultures or animal models) arereproducible in human subjects. Optimizing new production andpreservation methods for these novel strains is critical to evaluatethem towards improving human/animal health. Our laboratory has isolateda new strain of L. johnsonii (N6.2) (Lai et al. 2009) that mitigated theonset of diabetes type one in the genetically predisposed BioBreedinganimal model (Valladares et al 2010). Paradoxically, one of themechanisms involved in modulation of host response is through theproduction of reactive oxygen species (ROS) (Valladares et al. 2013). Wedetermined that L. johnsonii N6.2 is able to produce 1-120 in the hostgastrointestinal system and large amounts when suspended in salinephosphate buffer during the biomass production steps (Valladares et al.2015). Thus, designing effective methods to produce and preserve L.johnsonii N6.2 biomass while maintaining the ability to efficientlygenerate H₂O₂ as a mediator in the host represents an importantchallenge.

The oxidative stress conditions during industrial manufacturing wouldrepresent a large contributor to the survival of L. johnsonii N6.2.Based on our analysis, we hypothesized that the addition of a naturalcompound with antioxidant activity may improve the survival of thisstrain. In addition, L. johnsonii N6.2 has the ability to produceesterases, releasing esterified phytophenols and increasing their redoxquenching activity (Lai et al. 2009). In this work, we describe the useof blueberry aqueous extracts (BAE) as a phytophenol source to enhanceL. johnsonii N6.2 survival during culturing and cell harvesting.Blueberries contain natural compounds with high antioxidant activity dueto the high content and diversity of soluble phenolic compounds(Correa-betanzo et al. 2014; Correa-Betanzo et al. 2015).

The results of the Examples disclosed herein demonstrates that L.johnsonii N6.2 tolerance to industrial processing stressors such asaeration (oxidative stress), lyophilization (freeze-drying) andshelf-life is improved by utilizing natural antioxidants duringproduction.

Definitions

As used herein, the verb “comprise” as is used in this description andin the claims and its conjugations are used in its non-limiting sense tomean that items following the word are included, but items notspecifically mentioned are not excluded. In addition, reference to anelement by the indefinite article “a” or “an” does not exclude thepossibility that more than one of the elements are present, unless thecontext clearly requires that there is one and only one of the elements.The indefinite article “a” or “an” thus usually means “at least one.”

As used herein, the term “subject” or “patient” refers to any vertebrateincluding, without limitation, humans and other primates (e.g.,chimpanzees and other apes and monkey species), farm animals (e.g.,cattle, sheep, pigs, goats and horses), domestic mammals (e.g., dogs andcats), laboratory animals (e.g., rodents such as mice, rats, and guineapigs), and birds (e.g., domestic, wild and game birds such as chickens,turkeys and other gallinaceous birds, ducks, geese, and the like). Insome implementations, the subject may be a mammal. In otherimplementations, the subject may be a human.

The terms “pre-diabetic,” “pre-diabetes,” “pre-diabetic state,” or “pretype-1 diabetes,” as used herein refers to a subject at risk fordiabetes, and in particular, a subject at risk for type-1 diabetes. Forexample, a pre-diabetic patient or subject may have a fasting bloodsugar level between 100 and 125 mg/dL.

The term “diabetic state,” or “diabetes” as referred to herein refers toa state in which the blood sugar level reaches at least 126 mg/dL, inone example. Symptoms of Type 1 diabetes include frequent urination,excess thirst, weight loss (often sudden), skin infections,bladder/vaginal infections, and abdominal pain. Diabetes may bediagnosed with a blood test, which measures blood sugar level, and alsomeasures levels of insulin and antibodies to confirm the diagnosis.

As used herein, by the term “effective amount,” “amount effective,”“therapeutically effective amount,” or the like, it is meant an amounteffective at dosages and for periods of time necessary to achieve thedesired result. These terms refers to an amount of an enumerated agent,which, when administered or co-administered in a proper dosing regimen,is sufficient to reduce or ameliorate the severity, duration, orprogression of the disorder being treated (e.g., type 1 diabetes),prevent the advancement of the disorder being treated (e.g., type 1diabetes), cause the regression of the disorder being treated (e.g.,type 1 diabetes), or enhance or improve the prophylactic or therapeuticeffects(s) of another therapy. The full therapeutic effect does notnecessarily occur by administration of one dose and may occur only afteradministration of a series of doses. Thus, a therapeutically effectiveamount may be administered in one or more administrations per day forsuccessive days.

As used herein, the term “plant extract” refers to a substance derivedfrom a plant source that naturally contains phenolic compounds,including extracts prepared from the whole plant or from various partsof the plant, such as the fruits, leaves, stems, roots, bark, etc. Thus,the method of this invention is not limited to the particular part ofthe plant used to prepare the extract. The present method can use anysource of anthocyanins, proanthocyanidins and/or flavanoids, mosttypically from botanically derived plant materials such as seeds,fruits, skins, vegetables, nuts, tree barks, and other plant materialsthat contain phenolic compounds. Most colored fruits, berries, andvegetables are known to contain phenolic compounds. Examples of plants,fruits, berries, and vegetables that contain phenolic compounds include,but are not limited to, blueberries, bilberries, elderberries, plums,blackberries, strawberries, red currants, black currants, cranberries,cherries, raspberries, grapes, currants, hibiscus flowers, bell peppers,beans, peas, red cabbage, purple corn, and violet sweet potatoes. Theraw plant material may be used either as is (wet) or may be dried priorto extraction. Optionally, the raw plant material may be presorted byseparating and removing the components low in anthocyanins,proanthocyanidins, and/or flavanoids prior to extraction. The plantextract may be an extract in powdered form and reconstituted in anaqueous solution. In a specific example, the extract is blueberryaqueous extract (BAE) comprised of a blueberry powder dissolved indeionized water, centrifuged and filtered.

The term Lactobacillus sp. refers to Lactobacillus johnsonii andLactobacillus reuteri. In a specific embodiment, Lactobacillus johnsoniipertains to a specific strain L. johnsonii N6.2. A culture ofLactobacillus johnsonii N6.2 has been deposited with the American TypeCulture Collection (ATCC), 10801 University Blvd., Manassas, Va.20110-2209 USA. The deposit has been assigned accession number ATCC No.PTA-122064 by the repository and was deposited on Mar. 19, 2015. Thesubject culture has been deposited under conditions that assure thataccess to the culture will be available during the pendency of thispatent application to one determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 CFR 1.14 and 35 U.S.C. 122.The deposit is available as required by foreign patent laws in countrieswherein counterparts of the subject application, or its progeny, arefiled. However, it should be understood that the availability of adeposit does not constitute a license to practice the subject inventionin derogation of patent rights granted by governmental action.

Further, the subject culture deposit will be stored and made availableto the public in accord with the provisions of the Budapest Treaty forthe Deposit of Microorganisms, i.e., it will be stored with all the carenecessary to keep it viable and uncontaminated for a period of at leastfive years after the most recent request for the furnishing of a sampleof the deposit, and in any case, for a period of at least 30 (thirty)years after the date of deposit or for the enforceable life of anypatent which may issue disclosing the culture. The depositoracknowledges the duty to replace the deposit should the depository beunable to furnish a sample when requested, due to the condition of thedeposit. All restrictions on the availability to the public of thesubject culture deposit will be irrevocably removed upon the granting ofa patent disclosing it.

As used herein, the terms “phenol(s)” and “phenolic compound(s)” areused interchangeably and include monomeric, oligomeric and polymericcompounds having one or more phenolic groups, and include, but are notlimited to, anthocyanins, proanthocyanidins, and flavonoids.

As used herein, the term “phenol-enriched composition” refers to acomposition enriched in one or more phenolic compounds and havingsubstantially depleted levels of non-phenolic compounds present in crudeextracts of plants, fruits, berries, and vegetables. Examples of suchnon-phenolic compounds include, but are not limited to, sugars,cellulose, pectin, amino acids, proteins, nucleic acids, plant sterols,fatty acids, and triglycerides. In one embodiment, the phenol-enrichedcompositions are obtained by obtaining a plant extract from one or moreberries and/or fruits containing phenolic compounds including, but notlimited to, blueberries, bilberries, elderberries, plums, blackberries,strawberries, red currants, black currants, cranberries, cherries,raspberries, and grapes and subjected the plant extract to a method ofseparating out the phenolic compounds from non-phenolic compounds.

The compositions disclosed herein may be administered to treat otherautoimmune related disorders, including, but not limited to, rheumatoidarthritis, multiple sclerosis, thyroiditis, inflammatory bowel disease,Addison's disease, pancreas transplantation, kidney transplantation,islet transplantation, heart transplantation, lung transplantation, andliver transplantation. The compositions may be administered to improveoverall gastric health, i.e., as a probiotic to reduce indigestion,abdominal pain and cephalic syndrome. U.S. Pat. Nos. 9,987,313 and9,474,773 and PCT Pub. WO2018/112465 disclose various uses forLactobacillus johnsonii, which are incorporated herein by reference.Those skilled in the art would appreciate that the new compositionsdescribed herein could be used for above note disorders and in place ofthe compositions recited in the cited patent references.

The terms “treat”, “treating” or “treatment of” as used herein refers toproviding any type of medical management to a subject. Treatingincludes, but is not limited to, administering a composition to asubject using any known method. for purposes such as curing, reversing,alleviating, reducing the severity of, inhibiting the progression of, orreducing the likelihood of a disease, disorder, or condition or one ormore symptoms or manifestations of a disease, disorder or condition.

The Lactobacillus sp bacteria useful in the disclosed composition may beprovided as a live culture, as a dormant material or a combinationthereof. Those skilled in the art will appreciate that the Lactobacillussp bacteria may be rendered dormant by, for example, a lyophilizationprocess, as is well known to those skilled in the art.

An example of an appropriate lyophilization process may begin with amedia carrying appropriate Lactobacillus sp bacteria to which an amountof a phenolic compound may be added for cell protection prior tolyophilization. Examples of phenolic compounds may be provided as anextract from a natural plant source such as a berry (e.g. blueberry). Ina further embodiment, an adjunct lyophilization protectant is combinedwith the Lactobacillus sp and phenolic compound prior to lyophilization.Examples of adjunct lyophilization protectants include, but are notlimited to, distilled water, polyethylene glycol, sucrose, trehalose,skim milk, xylose, hemicellulose, pectin, amylose, amylopectin, xylan,arabinogalactan, starch (e.g., potato starch or rice starch) andpolyvinylpyrrolidone. Gasses useful for the lyophilization processinclude but are not limited to nitrogen and carbon dioxide.

Compositions

In one aspect, the Lactobacillus sp. bacteria in the disclosedcomposition may be provided as a dispersion in a solution or media. Inanother aspect, the Lactobacillus sp bacteria in the disclosedcomposition may be provided as a semi-solid or cake. In another aspect,the Lactobacillus sp. bacteria in the disclosed composition may beprovided in powdered form.

The disclosed compositions may be packaged in pharmaceuticallyacceptable vehicle, such as capsules, suppositories, tablets, food/drinkand the like. Optionally, the disclosed Lactobacillus/phenol mixture orlyophilized mixture may include various pharmaceutically acceptableexcipients, such as microcrystalline cellulose, mannitol, glucose,defatted milk powder, polyvinylpyrrolidone, starch and combinationsthereof.

In one aspect, the disclosed compositions may be prepared as a capsule.The capsule (i.e., the carrier) may be a hollow, generally cylindricalcapsule formed from various substances, such as gelatin, cellulose,carbohydrate or the like. The capsule may receive theLactobacillus/phenol mixture or lyophilized mixture therein. Optionally,and in addition to the appropriate Lactobacillus/phenol mixture orlyophilized mixture, the capsule may include but is not limited tocoloring, flavoring, rice or other starch, glycerin, caramel colorand/or titanium dioxide.

In another aspect, the Lactobacillus/phenol mixture or lyophilizedmixture may be prepared as a suppository. The suppository may includebut is not limited to the appropriate Lactobacillus/phenol mixture orlyophilized mixture and one or more carriers, such as polyethyleneglycol, acacia, acetylated monoglycerides, carnuba wax, celluloseacetate phthalate, corn starch, dibutyl phthalate, docusate sodium,gelatin, glycerin, iron oxides, kaolin, lactose, magnesium stearate,methyl paraben, pharmaceutical glaze, povidone, propyl paraben, sodiumbenzoate, sorbitan monoleate, sucrose talc, titanium dioxide, white waxand coloring agents.

In a further aspect, the Lactobacillus/phenol mixture or lyophilizedmixture may be prepared as a tablet. The tablet may include theappropriate Lactobacillus/phenol mixture or lyophilized mixture and oneor more tableting agents (i.e., carriers), such as dibasic calciumphosphate, stearic acid, croscarmellose, silica, cellulose and cellulosecoating. The tablets may be formed using a direct compression process,though those skilled in the art will appreciate that various techniquesmay be used to form the tablets.

In yet another aspect, the disclosed composition may be formed as foodor drink or, alternatively, as an additive to food or drink, wherein anappropriate quantity of Lactobacillus/phenol mixture or lyophilizedmixture is added to the food or drink to render the food or drink thecarrier.

The concentration of the Lactobacillus sp. bacteria in the disclosedcomposition may vary depending upon the desired result, the type ofbacteria used, the form and method of administration, among otherthings. For example, a composition may be prepared having a count ofLactobacillus sp. bacteria in the preparation of no less than about1×10⁶ colony forming units per gram, based upon the total weight of thepreparation.

Alternatively, one or more other excipients may be included in thecomposition to (1) impart satisfactory processing and compressioncharacteristics to the composition (e.g., adjust the flowability,cohesion and other characteristics of the composition) and (2) giveadditional desirable physical characteristics to the tables (e.g. color,stability, hardness, disintegration). Mostly the excipients aid in thedelayed release of the drug from the composition to achieve regionaldelivery to the lower GI. As used herein, the term “excipient” mayinclude all excipients present in the dosage form, including allcomponents other than the drug entity and the hydrocolloid gum fromhigher plants. A plurality of excipient substances may be present in anydosage form, and may include multiple substances having similarpharmaceutical function (e.g., lubricants, binders, diluents) or similarstructure (e.g., a mixture of monosaccharides). Preferably the fewerexcipients present the better. Such excipients are present in an amountsufficient to provide the composition with the desired delayedrelease/regional delivery characteristics, hardness rating and handlingcharacteristics and will generally be present at a level of about 2% byweight to about 50% by weight, preferably about 2% by weight to about40% by weight and more preferably about 2% to about 10% by weight.Excipients may be selected from many categories known in thepharmaceutical arts. The excipients used will be chosen to achieve thedesired object of the invention keeping in mind the activity of the drugbeing used, as well as its physical and chemical characteristics such aswater solubility and possible interactions with the excipients to beused.

For example with drugs that are more water soluble, generally a lowerpercentage by weight of excipients will be used, i.e., less than about20% or from about 2% to about 15% by weight, preferably no more thanabout 10% by wt, while for drugs that are less water soluble a higherpercentage by weight may be used, e.g., about 20% up to about 40% by wt.These levels may be adjusted to achieve the desired hardness andporosity of the final tablet composition to obtain the delayed releaseprofile.

From the foregoing discussion, it is seen that one aspect of thisinvention is a particle mass of a solid dosage form that can beadministered orally as a tablet. Thus, the composition is neither aliquid nor a gas, but a solid tablet having an amount of drug as a unitdosage. Generally, this unit dosage will be an amount that can beswallowed by a human subject and may vary from a total of about 100milligrams to about 1500 mg, preferably no more than about 1200 mg andparticularly no more than about 800 mg. For children, the size of thetablet may be significantly less than for adults, and for elderlypatients who have difficulty swallowing, the total amount may be lessthan what would be viewed as a normal amount for adults. It is to beunderstood that the tablets of this invention may be designed as asingle tablet having a unit dosage amount or several smaller tablets,e.g. 2-5, may be combined in a capsule for oral administration. Thecomposition used to prepare the tablet may be granulated.

In a typical embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted for oraladministration to humans.

The amount of the Lactobacillus sp. which will be effective in thetreatment of a particular disease or disorder will depend on the natureof the disease or disorder, and can be determined by standard clinicaltechniques. In addition, in vitro and in vivo assays may optionally beemployed to help identify optimal dosage ranges. The dosage will dependon the body weight of the subject. However, in one example suitabledosage ranges for oral administration or parenteral administration maybe about 10 pg to 100 mg, 20 pg to 50 mg, 0.1 mg to 20 mg, or 0.5 mg to10 mg (calculated either per kg body weight or as total dose perindividual). Suitable dosage ranges for intranasal administration aregenerally about 0.01 pg/kg body weight to 1 mg/kg body weight. Effectivedoses may be extrapolated from dose-response curves derived from invitro or animal model test systems. Suppositories generally contain anactive ingredient in the range of 0.5% to 10% by weight; oralformulations preferably contain 10% to 95% active ingredient.

The general approaches to delivering drugs to the lower GI tract (e.g.small intestine and colon) for interaction with immune cells in themucosa of the lower GI tract include: 1) enteric coating designed torelease drug in the more alkaline environment of the gastrointestinaltract, 2) bioerodible coatings and matrices, 3) prodrugs, 4)timed-release systems and, 5) enteric polymeric material-based releasesystems that release drug after they transit through the stomach andreach the intestines. A general discussion of these approaches andothers may be found in PCT Patent application No. PCT/US91/03014 bySintov and Rubinstein.

Process of Preparation

Capsules containing Lactobacillus sp. can be prepared according to knowntechniques. See for example US Pat. Pub. 20170368049 incorporated byreference. For preparing solid compositions such as tablets, theprincipal active ingredient may be mixed with a pharmaceutical carrier,e.g. conventional tableting ingredients such as corn starch, lactose,sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalciumphosphate or gums, and other pharmaceutical diluents, e.g. water. Thetablets or pills of the novel composition can be coated or otherwisecompounded to provide a dosage form affording the advantage of prolongedaction. For example, the tablet or pill can comprise an inner dosage andan outer dosage component, the latter being in the form of an envelopeover the former. The two components can be separated by an enteric layerwhich serves to resist disintegration in the stomach and permits theinner component to pass intact into the intestine or lower GI tract, orto otherwise be delayed in release. A variety of materials can be usedfor such enteric layers or coatings, such materials including a numberof polymeric acids and mixtures of polymeric acids with such materialsas shellac, acetyl alcohol and cellulose acetate.

In preparing the tablet compositions of this invention one may usepharmaceutical compression or molding techniques, preferably the formerdue to its adaptability to large scale production methods. Usingtechniques known in the art, the tablets of the invention may take anyappropriate shape such as discoid, round, oval, oblong, cylindrical,triangular, hexagonal, and the like. The tablets may be coated oruncoated. If coated they may be sugar-coated (to cover objectionabletastes or odors and to protect against oxidation), film coated (a thinfilm of water soluble matter for similar purposes), or enteric coated(to resist dissolution in gastric fluid but allow disintegration of thecoating in the small intestine—as discussed herein before). Depending onwhether the tablet is a uniform matrix tablet, an active core tablet ora concentration gradient tablet, the process for preparation will varyslightly.

In order to ensure tablet hardness and uniformity of weight, content andother items, it is preferable to prepare the tablets having thecomposition of this invention by using a pre-granulation technique. Ingeneral, the granulation techniques can include the wet granulationmethod, the fluid bed granulation method, the dry granulation method ordirect compression.

Once the tablets are appropriately formed, they can then be coated byany of the necessary coating techniques as discussed in Chapter 90 ofRemington's. For example, the tablets may be sugar-coated in accordancewith the procedure discussed therein or film coated or preferablyenterically coated. Enteric coating is preferred in the tablets of thisinvention to minimize the release of any of the drug in the upper GI andassure the release to the lower GI particularly the colon. As much aspertinent of the Remington's sections of Chapters 88 and 90 isincorporated herein by reference.

Administration

In some embodiments, the composition embodiments comprisingLactobacillus sp. described herein will be administered orally to amammalian subject in need thereof using a level of pharmaceuticalcomposition that is sufficient to provide the desired physiologicaleffect. The mammalian subject may be a domestic animal or pet butpreferably is a human subject. The level of pharmaceutical compositionneeded to give the desired physiological result is readily determined byone of ordinary skill in the art. Other parameters that may be takeninto account in determining dosage for the pharmaceutical compositionembodiments described herein may include disease state of the subject orage of the subject.

The compositions may take the form of suspensions, solutions oremulsions in oily or aqueous vehicles and may contain formulatory agentssuch as suspending, stabilizing and/or dispersing agents. In someembodiments, the composition embodiments described herein may beadministered orally or intravenously via parenteral nutritional therapyto a subject via an emulsion. The emulsion may include, in someembodiments, an aqueous continuous phase and a dispersed phase. Theboundary between the phases called the “interface”. The presentemulsions are adapted for application to a mucosal surface of avertebrate animal, preferably a mammal, including humans. Thesecompositions improve the permeability and bioavailability of activecompounds after application to a mucous surface. Mucosal surfaces ofinterest include the intestinal mucosa. Use of bioadhesive polymers inpharmaceutical emulsions affords enhanced delivery of drugs inbioadhesive polymer-coated suspensions, in some examples. Bioadhesivepharmaceutical emulsions may be used to deliver the Lactobacillus sp.described herein to: a) prolong the residence time in situ, therebydecreasing the number of drug administrations required per day; and b)may be localized in the specified region to improve and enhancetargeting and bioavailability of delivered drugs.

The ability to retain and localize a drug delivery emulsion in aselected region leads to improved bioavailability, especially for drugsexhibiting a narrow window of adsorption due to rapid metabolic turnoveror quick excretion. Intimate contact with the target absorption membraneimproves both the extent and rate of drug absorption.

Bioadhesion is the characteristic of certain natural and syntheticpolymers of binding to various biological tissues. Of particularinterest are polymers which bind to the mucous lining that covers thesurface of many tissues which communicate directly or indirectly withthe external environment, such as the gut, for example. Mucus bindingpolymers may be referred to as mucoadhesive. Several bioadhesive, andspecifically mucoadhesive, polymers are known. The chemical propertiesof the main mucoadhesive polymers are summarized as follows:

-   -   a. strong H-bonding groups (—OH, —COOH) in relatively high        concentration;    -   b. strong anionic charges;    -   c. sufficient flexibility of polymer backbone to penetrate the        mucus network or tissue crevices;    -   d. surface tension characteristics suitable for wetting mucus        and mucosal tissue surfaces; and    -   e. high molecular weight.

Bioadhesive polymers may be used in the pharmaceutical compositionembodiments described herein, examples of bioadhesive polymers currentlyused in pharmaceutical preparations include: carboxymethylcellulose(CMC), hydroxypropylmethylcellulose (HPMC), polyacrylic andpolymethacrylic acid and their derivatives, pectin, alginic acid,chitosan, polyvinylpyrrolidone, hyaluronic acid, and polyvinyl alcohol.The most frequently used polymer is Carbopol (Carbomer), which is a highmolecular weight polyacrylic acid polymer. It is used in manyformulations for bioadhesive drug delivery systems, as a suspendingagent, as a tablet coating, and in ocular suspensions.

Pharmaceutical composition embodiments described herein may include thecomposition comprising a Lactobacillus/phenol lyophilized mixture.incorporated into inert lipid carriers such as oils, surfactantdispersions, emulsions, liposomes etc. Self-emulsifying formulations areideally isotropic mixtures of oils, surfactants and co-solvents thatemulsify to form fine oil in water emulsions when introduced in aqueousmedia. Fine oil droplets would pass rapidly from stomach and promotewide distribution of drug throughout the GI tract, thereby overcome theslow dissolution step typically observed with solid dosage forms. Theseembodiments may provide control release self-emulsifying pellets,microspheres, tablets, capsules etc. that increase the use of“self-emulsification.”.

In further embodiments, Lactobacillus/phenol lyophilized mixturecomposition embodiments described herein may be microencapsulated fordelivery to a subject. Microencapsulation (ME) offers the potential toreduce the adverse effects on probiotic viability in thegastrointestinal (GI) tract environment. ME separates microorganismcells from their environment until they are released. Controlled releaseof the Lactobacillus is a particular benefit of ME. It is beneficial forencapsulated probiotic microorganisms to be released in the smallintestine where the Peyer's patches exist to activate the immune system,in some embodiments.

Oral delivery will be the most straightforward mode of administration todeliver the Lactobacillus sp. to the gut mucosa. However, in alternativeembodiments, other methods of administration are contemplated.Accordingly, suitable methods for administering a Lactobacillus sp.containing composition (e.g., Lactobacillus/phenol lyophilized mixture)in accordance with the methods of the presently-disclosed subject matterinclude, but are not limited to, systemic administration, parenteraladministration (including intravascular, intramuscular, and/orintraarterial administration), oral delivery, buccal delivery, rectaldelivery, subcutaneous administration, intraperitoneal administration,inhalation, dermally (e.g., topical application), intratrachealinstallation, surgical implantation, transdermal delivery, localinjection, intranasal delivery, and hyper-velocityinjection/bombardment. Where applicable, continuous infusion can enhancedrug accumulation at a target site (see, e.g., U.S. Pat. No. 6,180,082).In some embodiments of the therapeutic methods described herein, thetherapeutic compositions are administered orally, intravenously,intranasally, or intraperitoneally to thereby treat a disease ordisorder.

Regardless of the route of administration, the compositions of thepresently-disclosed subject matter typically not only include aneffective amount of a Lactobacillus sp. but are typically administeredin amount effective to achieve the desired response. As such, the term“effective amount” is used herein to refer to an amount of thetherapeutic composition (e.g., a Lactobacillus sp., and apharmaceutically vehicle, carrier, or excipient) sufficient to produce ameasurable biological response (e.g., a decrease in diabetes symptoms orincrease in immune function). Actual dosage levels of active ingredientsin a therapeutic composition of the present invention can be varied soas to administer an amount of the active compound(s) that is effectiveto achieve the desired therapeutic response for a particular subjectand/or application. Of course, the effective amount in any particularcase will depend upon a variety of factors including the activity of thetherapeutic composition, formulation, the route of administration,combination with other drugs or treatments, severity of the conditionbeing treated, and the physical condition and prior medical history ofthe subject being treated. Preferably, a minimal dose is administered,and the dose is escalated in the absence of dose-limiting toxicity to aminimally effective amount. Determination and adjustment of atherapeutically effective dose, as well as evaluation of when and how tomake such adjustments, are known to those of ordinary skill in the art.

Examples

L. johnsonii N6.2 Growth Conditions

L. johnsonii N6.2 was routinely grown in MRS broth (Remel, Lenexa, KS,USA). MNS media is a modified MRS broth without glucose, formulated asfollows: peptone 10 g, meat extract powder 10 g, yeast peptone 5 g,K₂HPO₄ 2 g, sodium acetate 5 g, ammonium citrate tribasic 2 g,MgSO₄·7H₂O 0.2 g, MnSO₄·H₂O 0.05 g, and Tween-80 1 g raised to a finalvolume of 1 L with distilled water, pH=6.5±0.2. To prepare blueberryenriched media (MNS-BB), the blueberry powder (Tifblue/Rubel 50/50blend, US Highbush Blueberry Council, Folsom, California, USA), wasdissolved in deionized water (at 1, 5 or 10% w/v), stirred for 60 min,and then centrifuged at 12000 rpm for 30 min at 4° C. The pellet wasdiscarded and the supernatant filtered (Whatman N° 1 filter paper). Theresulting BAE was used to solubilize the components of the MNS media.The resulting solubilizations constitute MNS-BB1, MNS-BB5, and MNS-BB10in accordance with the different blueberry concentrations. MNS-GF is MNSmedia supplemented with 3% w/v of glucose and 3% w/v of fructose. Allculture media were sterilized by filtration (0.22 μm).

To prepare the inoculum, L. johnsonii N6.2 culture grown for 16 h at 37°C. without agitation was inoculated at an initial OD=0.05. When aerobicconditions were used, the cultures were incubated in a shaker at 100 rpmat 37° C. Cell growth was monitored by periodic measurements of theculture's optical density at 600 nm (OD₆₀₀). Colony forming units(CFU/ml) were determined by performing serial dilutions with peptonewater (peptone extract 0.9% w/v). Subsequently, the cell suspensionswere plated on MRS agar plates. Plates were incubated at 37° C. for 48 hunder microaerophilic conditions.

Determination of H₂O₂

The production of H₂O₂ was determined by using colorimetric stripsfollowing the manufacturer's instructions (Quantofix, Macherey-Nagel,Germany). Alternatively, Amplex, Red Hydrogen peroxide/peroxidase assaykit (Invitrogen, CA, USA) and 4-aminoantipyrine and phenol (AAP) wereused (Marty-Teysset et al. 2000).

Phytophenol Processing and Recovery

Residual phenolic in growth media (MNS-GF or MNS-BB10) were recovered asfollows: samples of the growth media were taken at different time pointsalong L. johnsonii N6.2 growth kinetics. The supernatant was recoveredby centrifugation (4000 g for 30 min at 4° C.) and saved as aliquots at−20° C. until processing. Soluble phenolic compounds were recovered bysolid-phase extraction using a C18 HYPERSEP column (ThermoScientific,WI, USA) as described by Correa-Betanz et al. (2015).

Determination of Total Polyphenols

The total polyphenol content in MNS-GF or MNS-BB10, as well as in thepurified fractions were quantified using Folin-Ciocalteau's reagentaccording to Sanchez-Rangel et al. (2013) with slight modifications.Briefly, the samples (15 μL) were diluted in 240 μL of distilled waterin a 96-well microplate. Then, 15 μL of the Folin-Ciocalteau's phenolreagent, 2N (Sigma-Aldrich, USA) was added and the mixtures wereincubated 5 min at room temperature. The characteristic blue color wasdeveloped by adding 30 μL of 5% Na₂CO₃ solution. The plates wereincubated in the dark for 2 h at room temperature before measuring theabsorbance. The absorbance of the samples was measured at 725 nm using aSinergy HT microplate reader (Biotek, Winooski, VT, USA). The totalphenolic concentration was estimated from a calibration curve usinggallic acid as standard control. The polyphenol concentration wasexpressed as micrograms (μg) of gallic acid equivalent per mL of sample.

Radical Scavenging Activity

The total scavenging activity of the culture media was assessed by usingthe DPPH method described by Dong et al. (2017). The samples (MNS-GF,MNS-BB10 or its polyphenol enriched fractions) were diluted (½ and 1/10)in methanol. The reaction mixture containing 20 μL of each sample and180 μL of 0.2 mM DPPH solution was incubated at room temperature for 30min. The quenching ability of L-ascorbic acid was used as a positivecontrol. The absorbance was measured at 515 nm using a Sinergy HTmicroplate reader (Biotek, Winooski, VT, USA). The scavenging activity(SA) was estimated by the following formula: %SA=[(Abs_(control-blank)-Abs_(sample-blank))/Abs_(control-blank)]×100.Total radical scavenging activity (% SA) is expressed in percent ofquenching activity per g of gallic acid equivalents.

Evaluation of Aqueous Blueberry Extract on the Survival of L. johnsoniiN6.2 to Freeze-Drying

The effect of different cryo/drying preservatives was tested on thetolerance of L. johnsonii N6.2 to freeze-drying. L. johnsonii N6.2 wasgrown in MNS-GF or MNS-BB10 media to stationary phase (24 h of growth),centrifuged (6000 rpm for 20 min at 4° C.) and the pellet obtained waswashed once with phosphate-buffered saline (PBS pH=7.4). The bacteriawere resuspended as a 10× concentrate in PBS with skim milk (10% w/v),whey protein concentrate and sodium alginate (WPC/SA, 20% and 1% w/vrespectively) or PBS-BB buffer (PBS supplemented with 1, 5 or 10%blueberry powder, solubilized and filtered as indicated above). Bacteriacells suspended in PBS were used as controls. Frozen samples maintainedat −80° C. for 24 h were lyophilized and the freeze-dried powder wasstored at 4° C. to evaluate further cell viability.

Results

L. johnsonii N6.2 Shows Reduced Survival During Stationary Phase ofGrowth.

L. johnsonii N6.2 growth and survival were evaluated by cell counts andexpressed as CFU/ml of culture media (FIG. 1 ). The cultures wereincubated with agitation (aerobic) and static (microaerophilic)conditions to compare aeration effects on cell growth and viability.Both growth conditions resulted in similar growth kinetics during thefirst 12 h of growth (OD₆₀₀>1.0). However, growth in aerobic cultureshalted thereafter showing a drastic drop in cell viability as wasdemonstrated by CFU counts (FIG. 1 a ). After 36 h of growth, thisculture showed a total decrease of 5-log units. The loss of cellviability correlated with a large accumulation of H₂O₂ measured in theculture media (880 μM of H₂O₂) (FIG. 1 a ).

In contrast, microaerophilic cultures showed that cell viabilitydecreased only by 1-log unit after 24 h of growth. The amount of H₂O₂ at36 h of growth did not increase beyond 88 μM. No significant differenceswere observed in pH when comparing both cultures. These results suggestthat L. johnsonii N6.2 growth and viability in MRS media is stronglyaffected by oxidative stress in the form of H₂O₂ rather than itsproduction of organic acids. In addition, this data supports ourprevious findings where L. johnsonii N6.2 is able to produce H₂O₂ whencells are aerobically suspended in PBS (Valladares et al. 2015).

Blueberry Aqueous Extract Improved L. johnsonii N6.2 Growth and Survivalto Aerobic Stress.

The previous results prompted us to evaluate bacterial growth in a mediasupplemented with antioxidants. Since blueberries have a highphytophenol content with high antioxidant potential (Correa-betanzo etal. 2014), the effect of total blueberry extract was tested on L.johnsonii N6.2 growth. The blueberry powder utilized in this studycontains 600 g/kg of total metabolizable sugars. The main carbohydrates,glucose and fructose, are presented in a 1:1 relationship. To performthe assays at a comparable concentration of sugars used in the standardMRS culture media, a new culture media without glucose was formulated(MNS). The MNS media was amended with increasing concentrations (1, 5 or10% w/v) of freeze-dried BB to reach a final sugar content of 6, 30 and60 g/L, named MNS-BB1, MNS-BB5 and MNS-BB10, respectively. The culturemedia used as a control (MNS-GF) was formulated by adding glucose andfructose in a 1:1 relationship in order for it to be comparable to thetotal metabolizable sugars found in the blueberry powder.

The first assay performed was to evaluate the effects of BAE on L.johnsonii N6.2 growth and cell survival in a microaerophilicenvironment. In the control media, MNS-GF, L. johnsonii N6.2 reached5×10⁷ CFU/ml after 12 h of growth (FIG. 1 b ). These cells maintainedfull viability during the first 24 h of growth, but decreased to 1×10⁶CFU/ml after 36 h of growth. The cultures formulated with 1% BAE did notinhibit growth, but rather the observed growth rates were comparable tothat obtained in MNS-GF. Interestingly, after 12 h of incubation themedia formulated with 5 and 10% BAE reached 1×10⁹ CFU/ml. When cellswere challenged to grow aerobically in MNS-BB10 (100 rpm), the highestcell count of 1×10⁹ was obtained after only 8 h of growth (FIG. 1 c ).This represents a significant improvement in biomass productivity whencompared to MRS. In addition, cell viability decreased only 1-log after36 h of growth and more importantly, the H₂O₂ remained lower than 80 μM(detection limit) under these conditions (FIG. 1 c ).

In the supernatants collected from this assay, polyphenols, soluble lowmolecular weight phenolic compounds, and total scavenging activity ofthe culture media were measured. Both aerobic and anaerobic conditionswere compared and the results are summarized in Table 1. The totalphenolic compounds (polyphenols+soluble free phenols) remained almostconstant during the first 12 h of growth with slight decrease after 24 hof growth. However, no significant differences were observed betweenboth conditions. Since reducing sugars, such as those found inblueberry, are able to interfere with the Folin-Coicalteu method indetermining total phenolic content, the phenolics were purified from thefermented media and quantified. Interestingly, we observed a steadyincrease in the purified phenolics isolated from the culture media inboth conditions in a growth dependent manner, where the free phenolicswere double the concentration determined at the beginning of the assay(See Table 1). The increase in free phenol fractions over time reflectsthe ability of L. johnsonii N6.2 to release esterified phenolics presentin the BAE, potentially, due to the enzyme activity of its cinnamoylesterases.

The total antioxidant activity was determined using the DPPH radicalscavenging (% SA) method. Scavenging activity (% SA) is expressed asreducing equivalents of 1 μg of gallic acid (GAE) determined from thephenolic content of each sample. No significant differences wereobserved comparing the growth conditions when the total polyphenolfraction was analyzed. However, significant changes were observed in thepurified free phenol fraction over time (Table 1). The increase in % SAwas independent of the growth condition. It was found that the quenchingactivity increased from % SA=68.4±10.3 to % SA=96.3±5.9 after 36 h ofmicroaerophilic growth conditions. Similar values were obtained for theaerobic cultures (% SA=72.3±3.6 to % SA=99.3±2.5). This reducing poweris equivalent to 2-3 μg of L-ascorbic acid (% SA=20.2±0.85). The resultssuggest that the increasing quenching ability of the culture media is adirect consequence of the cinnamoyl esterase of L. johnsonii N6.2previously described (Lai et al. 2009).

The Addition of Blueberry Aqueous Extract to the Growth Media ImprovedL. johnsonii N6.2 Survival to Freeze Drying.

The tolerance of L. johnsonii N6.2 to freeze-drying stress duringlyophilization was evaluated comparing cells cultured in MNS-GF andMNS-BB10. L. johnsonii N6.2 was grown in each media for 24 h understatic condition, washed, resuspended in PBS, freezed, and thensubjected to freeze-drying. No additional preservatives were used atthis step. Viable cell counts were determined at each step of theprocedure: namely before freezing at −80° C., after freezing at −80° C.for 24 h and immediately after the freeze-drying step. The cells grownin MNS-GF and in MNS-BB10 showed a similar tolerance to freezing stress(a decrease in 1.5- and 1-log in CFU/ml, respectively) while the cellsgrown in MNS-GF were significantly more sensitive to the drying step,decreasing 2.5-log units (FIG. 2 a ). Meanwhile, cells grown in MNS-BB10showed a decrease of only 1.4-log units (FIG. 2 a ). Aliquots of thefreeze-dried samples were maintained for 21 weeks at 4° C. These sampleswere used to evaluate the viability of freeze-dried cells afterrehydration. The results obtained suggest that cells grown in MNS-GFwere less viable (decreased 2-log units) than the cells collected fromMNS-BB10 cultures that only decreased in 1-log unit from Week 0 (FIG. 2b ). In summary, in absence of additional preservatives duringfreeze-drying, cells grown in MNS-BB10 showed increased resistance tothe lyophilization/storage process (a combined reduction in 3.5-log)when compared to MNS-GF cultures where we observed a reduction of6.0-log units when comparing to pre-freezing bacterial counts.

Blueberry Aqueous Extract is an Effective Protectant for Lyophilization.

Our next goal was to determine whether or not the addition of BAE to PBSsolution used to suspend the cells prior to the freeze-drying step wouldincrease the survival of L. johnsonii N6.2. Cultures grown in MNS-GF for24 h were washed and suspended in PBS containing increasingconcentrations of BAE to final concentrations of 1, 5 or 10% w/v(PBS-BB1, PBS-BB5 and PBS-BB10, respectively) (FIGS. 2 c and d ). It wasobserved that the survival of L. johnsonii N6.2 after freeze-dryingimproved with increasing concentrations of BAE (FIG. 2 c ). A 100%survival to freezing was observed in all conditions containing blueberry(PBS-BB1, PBS-BB5 or PBS-BB10). Interestingly, even the lowestconcentration of BAE was able to improve the survival to thefreeze-drying process. The cells preserved in PBS showed a drasticdecrease in viability, dropping 2.5 log units from the freezing tofreeze-drying step (FIG. 2 c ). In contrast, the survival to storage at4° C. was highly affected by the concentration of BAE. A steady decreasein survival was observed for cells lyophilized in either PBS or PBS-BB1,while cells in PBS-BB5 or PBS-BB10 resulted in a close to 100% survivalfor up to 21 weeks (FIG. 2 d ).

The effect of BAE was determined on L. johnsonii cultures grown underaerobic conditions (FIG. 3 ). Similarly to the cells grown under staticconditions, the combined use of BAE as a media supplement (MNS-BB10) oras a preservative (PBS-BB10) provided the maximal tolerance tolyophilization and subsequent storage at 4° C. (FIG. 3 ).

Blueberry as a Culture Additive Improves the Activity of Commonly UsedCryoprotectants.

Next, we analyzed the combined effect of BAE when it was added to thegrowth media and when used as an additive during lyophilization. Cellswere grown to stationary phase in either MNS-GF (FIGS. 4 a and b ) orMNS-BB10 (FIGS. 4 c and d ) and then lyophilized in the absence (PBS) orpresence of BAE (PBS-BB10). It was found that the use of BAE in both thegrowth media and as an additive resulted in maximal survival tofreeze-drying (FIGS. 4 a and c ).

The protective effect of the BAE was compared to other commonly usedprotectants during lyophilization such as 10% skim milk (PBS-milk) ormicroencapsulation using whey protein-sodium alginate (PBS-WPC/SA). Itwas found that PBS-milk and PBS-WPC/SA resulted in an almost 100%survival after freezing cells grown in either MNS-GF (FIG. 4 a ) orMNS-BB10 (FIG. 4 c ). For cells grown on MNS-GF, a significantdifference (p<0.039) was observed after the drying step among thepreservatives used, these being PBS-BB10>PBS-WPC/SA>PBS-milk (FIG. 4 a). A similar trend was observed in L. johnsonii N6.2 grown in MNS-BB10where the survival in PBS-Milk was slightly below the results obtainedby MNS-BB10 and PBS-WPC/SA (FIG. 4 c ). In all cases, the BAEdemonstrated to be a superior protectant to storage (FIGS. 4 b and d ).In the case of microencapsulated cells, the preservation was comparableonly when the cells were grown in presence of BAE (FIG. 4 d ).

Although the present invention has been described in considerable detailwith reference to certain preferred versions thereof, other versions arepossible. Therefore, the spirit and scope of the appended claims shouldnot be limited to the description of the preferred versions containedherein.

The reader's attention is directed to all papers and documents which arefiled concurrently with this specification and which are open to publicinspection with this specification, and the contents of all such papersand documents are incorporated herein by reference.

All the features disclosed in this specification (including anyaccompanying claims, abstract, and drawings) may be replaced byalternative features serving the same, equivalent or similar purpose,unless expressly stated otherwise. Thus, unless expressly statedotherwise, each feature disclosed is one example only of a genericseries of equivalent or similar features.

Any element in a claim that does not explicitly state “means for”performing a specified function, or “step for” performing a specificfunction, is not to be interpreted as a “means” or “step” clause asspecified in 35 U.S.C § 112, sixth paragraph. In particular, the use of“step of” in the claims herein is not intended to invoke the provisionsof 35 U.S.C § 112, sixth paragraph.

REFERENCES

-   Bhat, R., Suryanarayana, L. C., Chandrashekara, K. A., Krishnan, P.,    Kush, A., & Ravikumar, P. (2015). Lactobacillus plantarum mediated    fermentation of Psidium guajava L. fruit extract. Journal of    Bioscience and Bioengineering, 119(4), 430-432.    https://doi.org/10.1016/j.jbiosc.2014.09.007-   Bravo-ferrada, M., Brizuela, N., & Gerbino, E. (2015). Effect of    protective agents and previous acclimation on ethanol resistance of    frozen and freeze-dried Lactobacillus plantarum strains. Cryobiology    71, 522-528. https://doi.org/10.1016/j.cryobiol.2015.10.154-   Bustamante, M., Oomah, B. D., Rubilar, M., & Shene, C. (2017).    Effective Lactobacillus plantarum and Bifidobacterium infantis    encapsulation with chia seed (Salvia hispanica L.) and flaxseed    (Linum usitatissimum L.) mucilage and soluble protein by spray    drying. Food Chemistry, 216, 97-105.    https://doi.org/10.1016/j.foodchem.2016.08.019-   Correa-betanzo, J., Allen-vercoe, E., Mcdonald, J., Schroeter, K.,    Corredig, M., & Paliyath, G. (2014). Stability and biological    activity of wild blueberry (Vaccinium angustifolium) polyphenols    during simulated in vitro gastrointestinal digestion. FOOD    CHEMISTRY, 165, 522-531.    https://doi.org/10.1016/j.foodchem.2014.05.135-   Correa-Betanzo, J., Padmanabhan, P., Corredig, M., Subramanian, J.,    & Paliyath, G. (2015). Complex formation of blueberry (Vaccinium    angustifolium) anthocyanins during freeze-drying and its influence    on their biological activity. Journal of Agricultural and Food    Chemistry, 63(11), 2935-46. https://doi.org/10.1021/acs.jafc.5b00016-   Costa, M. G., Ooki, G. N., Vieira, A. D., Bedani, R., & Saad, S. M.    (2017). Synbiotic Amazonian palm berry (açai, Euterpe oleracea    Mart.) ice cream improved Lactobacillus rhamnosus GG survival to    simulated gastrointestinal stress. Food & Function, 8(2): 731-740.    doi: 10.1039/c6fo00778c.-   Dong, L.-M., Jia, X.-C., Luo, Q.-W., Zhang, Q., Luo, B., Liu, W.-B.,    . . . Tan, J.-W. (2017). Phenolics from Mikania micrantha and Their    Antioxidant Activity. Molecules (Basel, Switzerland), 22(7).    https://doi.org/10.3390/molecules22071140-   Endo, A., Teräsjärvi, J., & Salminen, S. (2014). Food matrices and    cell conditions influence survival of Lactobacillus rhamnosus GG    under heat stresses and during storage. International Journal of    Food Microbiology, 174, 110-2.    https://doi.org/10.1016/j.ijfoodmicro.2014.01.006-   Kechagia, M., Basoulis, D., Konstantopoulou, S., Dimitriadi, D.,    Gyftopoulou, K., Skarmoutsou, N., & Fakiri, E. M. (2013). Health    Benefits of Probiotics: A Review. ISRN Nutrition, 2013, 1-7.    https://doi.org/10.5402/2013/481651-   Klaenhammer, T. R., Kleerebezem, M., Kopp, M. V., & Rescigno, M.    (2012). The impact of probiotics and prebiotics on the immune    system. Nature Reviews Immunology, 12(10), 728-734.    https://doi.org/10.1038/nri3312-   Lai, K. K., Lorca, G. L., & Gonzalez, C. F. (2009). Biochemical    Properties of Two Cinnamoyl Esterases Purified from a Lactobacillus    johnsonii Strain Isolated from Stool Samples of Diabetes-Resistant    Rats. Applied and Environmental Microbiology, 75(15), 5018-5024.    http://doi.org/10.1128/AEM.02837-08-   Lorca, G. L., & Valdez, G. F. (2001). A low-pH-inducible,    stationary-phase acid tolerance response in Lactobacillus    acidophilus CRL 639. Current Microbiology, 42(1), 21-5.    https://doi.org/10.1007/s002840010172-   Marty-Teysset, C., de la Torre, F., & Garel, J. (2000). Increased    production of hydrogen peroxide by Lactobacillus delbrueckii subsp.    bulgaricus upon aeration: involvement of an NADH oxidase in    oxidative stress. Applied and Environmental Microbiology, 66(1),    262-7. doi: 10.1128/AEM.66.1.262-267.2000-   Nishiyama, K., Nakazato, A., Ueno, S., Seto, Y., Kakuda, T., Takai,    S., Yamamoto, Y., & Mukai, T. (2015). Cell surface-associated    aggregation-promoting factor from Lactobacillus gasseri SBT2055    facilitates host colonization and competitive exclusion of    Campylobacter jejuni. Molecular Microbiology, 98(4), 712-726. doi:    10.1111/mmi.13153.-   Nya, E. J. (2015). Development of Probiotics as Biotechnology-Driven    Product for Reducing the Incidence of Gastrointestinal Related    Disease, 4(9), 2013-2016.-   Rajam, R., Kumar, S. B., Prabhasankar, P., & Anandharamakrishnan, C.    (2015). Microencapsulation of Lactobacillus plantarum MTCC 5422 in    fructooligosaccharide and whey protein wall systems and its impact    on noodle quality. Journal of Food Science and Technology, 52(7),    4029-4041. https://doi.org/10.1007/s13197-014-1506-4-   Rodrigues, F. J., Omura, M. H., Cedran, M. F., Dekker, R. F.,    Barbosa-Dekker, A. M., & Garcia, S. (2017). Effect of natural    polymers on the survival of Lactobacillus casei encapsulated in    alginatemicrospheres. Journal of Microencapsulation, 34(5): 431-439.    doi: 10.1080/02652048.2017.1343872.-   Saarela, M., Virkajärvi, I., Nohynek, L., Vaari, A., & Mättö, J.    (2006). Fibres as carriers for Lactobacillus rhamnosus during    freeze-drying and storage in apple juice and chocolate-coated    breakfast cereals. International Journal of Food Microbiology,    112(2), 171-8. https://doi.org/10.1016/j.ijfoodmicro.2006.05.019-   Sánchez-Rangel, J. C., Benavides, J., Heredia, J. B.,    Cisneros-Zevallos, L., & Jacobo-Velázquez, D. A. (2013). The    Folin-Ciocalteu assay revisited: improvement of its specificity for    total phenolic content determination. Analytical Methods,    5(21), 5990. https://doi.org/10.1039/c3ay4l125 g-   Shiby, V. K., & Mishra, H. N. (2017). Fermented Milks and Milk    Products as Functional Foods—A Review Fermented Milks and Milk    Products as Functional Foods—A Review, 8398 (September).    https://doi.org/10.1080/10408398.2010.547398-   Valladares, R. B., Graves, C., Wright, K., Gardner, C. L., Lorca, G.    L., & Gonzalez, C. F. (2015). H 2 O 2 production rate in    Lactobacillus johnsonii is modulated via the interplay of a    heterodimeric flavin oxidoreductase with a soluble 28 Kd PAS domain    containing protein, 6 (July), 1-14.    https://doi.org/10.3389/fmicb.2015.00716-   Valladares, R., Bojilova, L., Potts, A. H., Cameron, E., Gardner,    C., Lorca, G., & Gonzalez, C. F. (2013). Lactobacillus johnsonii    inhibits indoleamine 2,3-dioxygenase and alters tryptophan    metabolite levels in BioBreeding rats. FASEB Journal, 27(4),    1711-1720. https://doi.org/10.1096/fj.12-223339-   Valladares, R., Sankar, D., Li, N., Williams, E., Lai, K. K.,    Abdelgeliel, A. S., . . . Lorca, G. L. (2010). Lactobacillus    johnsonii N6.2 mitigates the development of type 1 diabetes in BB-DP    rats. PLoS ONE, 5(5). https://doi.org/10.1371/journal.pone.0010507-   van de Guchte, M., Serror, P., Chervaux, C., Smokvina, T.,    Ehrlich, S. D., & Maguin, E. (2002). Stress responses in lactic acid    bacteria. Antonie van Leeuwenhoek, 82(1-4), 187-216.    http://dx.doi.org/10.1023/A:1020631532202-   Zheng, X., Fu, N., Duan, M., Woo, M. W., Selomulya, C., &    Chen, X. D. (2015). The mechanisms of the protective effects of    reconstituted skim milk during convective droplet drying of lactic    acid bacteria. Food Research International, 76, 478-488.    https://doi.org/10.1016/j.foodres.2015.07.045

What is claimed is:
 1. A method for treating or alleviating symptoms ofindigestion, abdominal pain or cephalic syndrome in a subject, acomposition comprising an effective amount of comprising at least oneLactobacillus sp. and at least one phenolic compound.
 2. The method ofclaim 1, wherein the at least one Lactobacillus sp. comprisesLactobacillus johnsonii.
 3. The method of claim 2, wherein Lactobacillusjohnsonii comprises Lactobacillus johnsonii N6.2.
 4. The method of claim1, wherein the at least one Lactobacillus sp. and at least one phenoliccompound are combined as mixture, and, optionally, wherein the mixtureis lyophilized.
 5. The method of claim 1, wherein administeringcomprises oral administration.
 6. A method of improving immune functionin a subject, comprising administering to the subject, a compositioncomprising an effective amount of comprising at least one Lactobacillussp. and at least one phenolic compound.
 7. The method of claim 6,wherein the at least one Lactobacillus sp. comprises Lactobacillusjohnsonii.
 8. The method of claim 7, wherein Lactobacillus johnsoniicomprises Lactobacillus johnsonii N6.2.
 9. The method of claim 6,wherein administering the composition increases expression of Toll likereceptor 7 (TLR7) and Toll like receptor 9 (TLR9) in the subject.
 10. Acomposition comprising at least one Lactobacillus sp. and at least onephenolic compound, the composition being produced by: mixing the atleast one at least one Lactobacillus sp. and at least one phenoliccompound to form a Lactobacillus/phenol mixture; and lyophilizing theLactobacillus/phenol mixture to form a Lactobacillus/phenol lyophilizedmixture.
 11. The composition of claim 10, wherein the at least onephenolic compound is present in a plant extract.
 12. The composition ofclaim 11, wherein the plant extract is a berry extract.
 13. Thecomposition of claim 12, wherein the berry extract is a dried powderthat reconstituted in an aqueous solution prior to the mixing step. 14.The method of claim 6, wherein administering comprises oral, intravenousor rectal administration.
 15. (canceled)
 18. (canceled)
 19. (canceled)